Production scale method of forming microparticles

ABSTRACT

The present invention relates to a method for forming microparticles of a material from microdroplets of a solution, wherein the solution comprises the material dissolved in a solvent. The method includes the steps of directing the microdroplets into a freezing zone, wherein the freezing zone is surrounded by a liquified gas, and wherein the microdroplets freeze. The frozen microdroplets are then mixed with a liquid non-solvent, whereby the solvent is extracted into the non-solvent, thereby forming the microparticles.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.10/006,991, filed on Dec. 6, 2001, now U.S. Pat. No. 6,726,860 which isa Continuation of U.S. patent application Ser. No. 09/587,821, filed onJun. 6, 2000, now U.S. Pat. No. 6,358,443 which is a Continuation ofU.S. patent application Ser. No. 09/305,413, filed on May 5, 1999, nowU.S. Pat. No. 6,153,129, which is a Continuation of U.S. patentapplication Ser. No. 08/443,726, filed on May 18, 1995, now U.S. Pat.No. 5,922,253, the entire teachings of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Many illnesses or conditions require a constant level of medicaments oragents in vivo to provide the most effective prophylactic, therapeuticor diagnostic results. In the past, medicaments were given in doses atintervals which resulted in fluctuating medication levels.

Attempts to control and steady medication levels have more recentlyincluded the use of many biodegradable substances, such as polymeric andprotein microspheres containing the medicament. The use of thesemicrospheres provided an improvement in the controlled release ofmedicaments by utilizing the inherent biodegradability of the polymer toimprove the release of the medicament and provide a more even,controlled level of medication.

However, many of these methods result in low yields of microspheres dueto a combination of the methods and apparatus used. Further, someprocesses cannot be scaled-up from experimental level to a commercialproduction level.

Therefore, a need exists for a method of forming microspheres with lowerlosses of biologically active agent, high product yields, andcommercial-scale feasibility.

SUMMARY OF THE INVENTION

This invention relates to a method for forming microparticles of amaterial from microdroplets of a solution, wherein the solutioncomprises the material dissolved in a solvent. The method includes thesteps of directing the microdroplets into a freezing zone, wherein thefreezing zone is surrounded by a liquified gas, and wherein themicrodroplets freeze. The frozen microdroplets are then mixed with aliquid non-solvent, whereby the solvent is then extracted into thenon-solvent, thereby forming the microparticles.

This invention has numerous advantages, for instance, this method andapparatus provides high yields, commercial production levels ofcontrolled release microparticles, an enclosed system for asepticprocessing, microparticle size control and process controlreproducibility

In addition, the method of invention permits greater tailoring oftemperature profiles during performance of the method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cut-away side elevational illustration of an apparatus ofthe invention suitable for forming microparticles of a material,according to the method of the invention by freezing microdroplets of asolution of the material in a solvent, within a freezing zone cooled byan encircling flow of liquified gas and then extracting the solvent fromthe frozen microdroplets, by exposure to a liquid non-solvent.

FIG. 2 is a cut-away side elevational illustration of another embodimentof an apparatus of the invention suitable for forming microparticles ofa material according to the method of the invention, by freezingmicrodroplets of a solution of the material in a solvent, within afreezing zone cooled by an encircling flow of a liquified gas and thenextracting the solvent from the frozen microdroplets, by exposure to aliquid non-solvent.

FIG. 3 is a cut-away side elevational illustration of yet anotherembodiment of an apparatus of the invention suitable for formingmicroparticles of a material according to the method of the invention,by freezing microdroplets of a solution of the material in a solvent,within a freezing zone cooled by an encircling flow of a liquified gasand then extracting the solvent from the frozen microdroplets, byexposure to a liquid non-solvent.

FIG. 4 is a cut-away side elevational illustration of an alternateembodiment of an apparatus of the invention suitable for formingmicroparticles of a material according to the method of the invention,by freezing microdroplets of a solution of the material in a solvent,within a freezing zone cooled by an encircling flow of a liquified gasand then extracting the solvent from the frozen microdroplets, byexposure to a liquid non-solvent.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method and apparatus for formingmicroparticles of a material from a solution of the material. Amicroparticle, as defined herein, comprises a particle of a materialhaving a diameter of less than about one millimeter. A microparticle canhave a spherical, non-spherical or irregular shape. It is preferred thata microparticle be a microsphere.

Materials suitable to form microparticles of this invention include, forexample, polymers, peptides, polypeptides, proteins, small moleculedrugs and pro-drugs.

A microparticle can also contain one or more additional substance, whichis dispersed within the microparticle. Wherein the material comprises apolymer, the polymer solution contains at least one biologically activeagent.

A biologically active agent, as defined herein, is an agent, or ametabolite of the agent, which possesses therapeutic, prophylactic ordiagnostic properties in vivo, in the form of said agent whenadministered, or after metabolism (e.g., a pro-drug, such ashydrocortisone succinate).

One embodiment of an apparatus of the invention, suitable for performingthe method of invention, is illustrated in FIG. 1. Said apparatusincludes vessel 10, typically in a cylindrical shape, having side wall12, vessel top 14, vessel bottom 16 and internal wall 18. Side wall 12and vessel bottom 16 are usually insulated, using conventionalinsulation methods, to minimize heat leakage from the outsideenvironment into vessel 10, thereby providing improved temperaturecontrol within vessel 10. Conventional insulation methods include, forexample, application of at least one layer of insulation material 17 tocover the outside surfaces of side wall 12 and vessel bottom 16. Othermeans of insulating include, for instance, vacuum jacketing side wall 12and vessel bottom 16 with radiation shielding. Suitable insulationmaterials include conventional insulation materials, such as mineralfiber, polystyrene, polyurethane, rubber foams, balsa wood or corkboard.

In this embodiment, vessel top 14 is typically not insulated, therebyallowing components of said apparatus, disposed at or near vessel top14, to be warmed by heat leakage into the vessel 10. Alternately, vesseltop 14 may also be insulated with a suitable insulation material.

Vessel 10 is fabricated with a material which can withstand conditionsduring steam sanitizing, of the inside of vessel 10, and can alsowithstand the temperatures and gas pressures experienced in vessel 10while performing the method of invention for forming microparticles 11.Suitable materials for vessel 10 include, for example, stainless steel,polypropylene and glass.

Vessel 10, in this embodiment, is a single unitary vessel, divided intofreezing section 20 and extraction section 22. Freezing section 20 isdisposed within, and substantially enclosed by, side wall 12, vessel top14 and internal wall 18. Extraction section 22 is disposed within, andsubstantially enclosed by, side wall 12, vessel bottom 16 and internalwall 18.

In an alternate embodiment, freezing section 20 and extraction section22 comprises separate vessels, wherein the freezing section vessel isdisposed generally above the extraction section vessel, and wherein thebottom of the freezing section vessel is connected to the top or to aside of the extraction section vessel.

Vessel 10 also includes means for directing liquified gas into freezingsection 20 to form liquified gas flow 24. Liquified gas flow 24 consistsof a spray of liquified gas and/or at least one stream of liquified gas.Liquified gas flow 24 begins in freezing section 20 at or near vesseltop 14, and then runs in a generally downward direction, toward internalwall 18. Within freezing section 20, at least a portion of liquified gasflow 24 runs substantially parallel with side wall 12. Liquified gasflow 24 is typically disposed at or near side wall 12. It is preferredthat side wall 12 is generally wetted by liquified gas flow 24.Furthermore, liquified gas flow 24 substantially encircles freezing zone26, which is approximately disposed about the radial centerline offreezing section 20. The extent to which liquified gas flow 24 has gapsin the encircling flow about freezing zone 26, is dependent upon thetype and number of liquified gas directing means employed.

At least one suitable liquified gas directing means is disposed at ornear vessel top 14, at a location which is radially displaced from thecenter of vessel top 14. The radial displacement of a liquified gasdirecting means, is sufficient if the liquified gas directing means doesnot significantly interfere with the formation of microdroplets 28, suchas by freezing a portion of a solution from which microdroplets 28 areformed at microdroplet forming means 30, thereby at least partiallyclogging microdroplet forming means 30. A liquified gas directing meanscan also interfere if a significant portion of microdroplets 28 impactsaid liquified gas directing means.

In the embodiment illustrated in FIG. 1, suitable liquified gasdirecting means include at least two spray nozzles 32 having a linedischarge or preferably a fan discharge (e.g., flood jet atomizer model1/8-K-SS-1, operated with a liquid gas pressure of about 20 psig; SpraySystems Co., Wheaton, Ill.), which are capable of spraying a liquifiedgas to form at least a portion of liquified gas flow 24. Spray nozzles32 are disposed in freezing section 20 at vessel top 14, and are aboutequidistantly spaced at positions approximately located on a circlecentered around the center of vessel top 14, or centered aroundmicrodroplet forming means 30 if radially displaced from said vessel topcenter. The number of spray nozzles 32 used will depend upon the arc ofthe nozzle discharge and the distance from nozzle 32 to the impact pointon side wall 12 of liquified gas flow 24.

With two spray nozzles 32 equidistantly displaced from the center of thetop of freezing section 20, encircling liquified gas flow 24 willtypically have two gaps about 180° apart, due to the usual inability ofspray nozzle 32 to spray in greater than a 180° arc. In a preferredembodiment, at least three spray nozzles are disposed in freezingsection 20 to form liquified gas flow 24 which encircles freezing zone26 typically without any significant gaps in the encircling flow.

Typically, three spray nozzles 32, equidistantly spaced will provide a360° liquified gas flow 24. In a more preferred embodiment, six spraynozzles are equidistantly disposed about the center of freezing section20.

A liquified gas directing means receives liquified gas, from at leastone liquified gas inlet 34. Liquified gas inlet 34 provides fluidcommunication between liquified gas source 36 and the liquified gasdirecting means. It is understood that other suitable liquified gasintroduction means, capable of directing liquified gas flow into theliquified gas directing means, can be used in place of, or incombination with, liquified gas inlet 34.

FIG. 2 illustrates another embodiment of a suitable liquified gasdirecting means of an apparatus of this invention. The apparatus of FIG.2 has many of the same elements of FIG. 1 and like elements aredesignated with like numerals. In said apparatus, suitable liquified gasdirecting means comprises weir 102 and liquified gas space 104. Weir 102is disposed within freezing section 20, between side wall 12 andfreezing zone 26. Weir 102 extends from internal wall 18, or alternatelyfrom side wall 12, and extends upwards toward vessel top 14. In oneembodiment, the top portion of weir 102 does not contact vessel top 14,thus permitting liquified gas to flow over the top of weir 102 andfurther into freezing section 20. Alternately, wherein weir 102 contactsvessel top 14, weir 102 is porous or slotted at the top of weir 102 (notshown) to permit liquified gas to flow through the top section of weir102, and further into freezing section 20.

Liquified gas space 104 is disposed within freezing section 20, betweenweir 102 and side wall 12. Liquified gas space 104 receives liquifiedgas from at least one liquified gas inlet 34. The liquified gas is thendirected over or through weir 102 further towards the center of freezingsection 20.

Referring back to FIG. 1, vessel 10 also includes microdroplet formingmeans 30, disposed in freezing section 20 at vessel top 14, for formingmicrodroplets 28 from a suitable solution. A microdroplet is definedherein as a drop of solution which, after freezing and subsequentextraction of the solution's solvent, will form a microparticle.Examples of suitable microdroplet forming means 30 include atomizers,nozzles and various gauge needles. Suitable atomizers include, forexample, external air (or gas) atomizers (e.g., Model SUE15A; SpraySystems Co., Wheaton, Ill.), internal air atomizers (e.g., SU12; SpraySystems Co.), rotary atomizers (e.g., discs, bowls, cups and wheels;Niro, Inc., Columbia, Md.), and ultrasonic atomizers (e.g., AtomizingProbe 630-0434; Sonics & Materials, Inc., Danbury, Conn.). Suitablenozzles include pressure atomization nozzles (e.g, Type SSTC Whirl JetSpray Drying Nozzles; Spray Systems Co., Wheaton, Ill.). Typical gaugesof needles used to form microdroplets 28 include needles with gaugesbetween about 16 and about 30.

In a preferred embodiment, microdroplet forming means 30 is an airatomizer, which can form microparticles 11 having a range of diametersbetween about 1 micrometer, or less, and about 300 micrometers. Averagemicroparticle size can be changed by adjusting the pressure of theatomizing gas, supplied to an air atomizer (e.g., nitrogen gas).Increased gas pressure results in smaller average microparticlediameters.

Microdroplet forming means 30 is fabricated from a material, orcombination of materials, which can withstand steam sanitizing and alsothe cold temperatures experienced in freezing section 20.

Microdroplet forming means 30 receives solution from at least onesolution inlet 38. Solution inlet 38 provides fluid communicationbetween solution source 40 and freezing section 20. It is understoodthat other suitable solution introduction means, such as a lance or another device capable of injecting a solution into a cold environment,can be used in place of, or in combination with, solution inlet 38.

Vessel 10 also includes at least one three-phase port 42, which isdisposed at internal wall 18, and provides fluid communication betweenfreezing section 20 and extraction section 22. Three-phase port 42 issized to allow the flow of a combination of frozen microdroplets 44,liquified gas and volatilized gas from freezing section 20 intoextraction section 22.

Extraction section 22 includes means for separating a liquified gas fromfrozen microdroplets 44. In one embodiment, a suitable separating meanscomprises a means for heating extraction section 22, which thenvolatilizes the liquified gas, thus separating it from frozenmicrodroplets 44, usually contained within the lower portion ofextraction section 22. Said heating means can also be used to warm thesolvent frozen within frozen microdroplets 44. Suitable means forheating can include heat leakage from the outside environment, throughside wall 12 and vessel bottom 16. Optionally, heating means caninclude, for example, electrical means such as heating coils, orrecirculating heat exchanger tubes 46, through which a fluid can becirculated to control temperature within extraction section 22 to firstvolatilize the liquified gas, and then subsequently warm the solvent infrozen microdroplets 44 to control solvent extraction rate.

An alternate separating means comprises filtered bottom tap 48, whichextends from the lower portion of extraction section 22. Filtered bottomtap 48, which contains filter 50, having a pore size less than thediameter of microparticles 11, typically ≦1 micrometer, is suitable forremoving liquids, such as liquified gas, from extraction section 22,while retaining frozen microdroplets 44, and possibly microparticles 11,within extraction section 22.

Gas outlet 52, which is disposed in extraction section 22 at internalwall 18, is suitable for directing gas, produced by volatilizingliquified gas, out of vessel 10. Gas outlet 52 can optionally include ameans for reducing pressure within vessel 10, for example a vacuumblower (e.g., CP-21 low temperature blower, Barber Nichols, Arvada,Colo.) or vacuum pump (e.g. E2M18 vacuum pump, Edwards High VacuumInternational, Crawley, West Sussex, England) suitable for impellinggases. Furthermore, gas outlet 52 typically includes filter 53 (e.g., a0.2 micrometer sterile filter) in the gas flow path to support anaseptic process and provide assurance that formed microparticles 11 meetsterility requirements.

Vessel 10 can optionally include gas outlets 52, disposed in extractionsection 22 and/or freezing section 20 (not shown). It is preferred thatno gas outlets be disposed in freezing section 20, as gas venting out offreezing section 20 can result in gas circulation currents which canreduce the yield of microparticles 11 produced.

In addition, vessel 10 can optionally include at least one overpressureprotection device (not shown), to protect the material integrity ofvessel 10 from overpressurization caused by the volatilization of aliquified gas. Typical overpressure protection devices include, forinstance, rupture disks or pressure relief valves.

Extraction section 22 also includes at least one non-solvent inlet 54,disposed at internal wall 18 and/or in side wall 12. Extraction section22 receives a liquid non-solvent from non-solvent inlet 54 in a streamor spray. Preferably, non-solvent in extraction section 22 formsextraction bath 56, which is disposed in at least the lower portion ofextraction section 22. Non-solvent inlet 54 provides fluid communicationbetween cold non-solvent source 58 and extraction bath 56. It isunderstood that other suitable means, for introducing a liquid into avessel under cold conditions, such as a lance or an other device capableof introducing a liquid under cold conditions, can be used in place of,or in combination with, non-solvent inlet 54.

In another embodiment, a suitable mixing means 60 for mixing frozenmicrodroplets 44 and non-solvent, is disposed in extraction bath 56.Mixing means 60 is provided to reduce the potential for formation ofextraction gradients within extraction bath 56, such as could occur iffrozen microdroplets 44 clumped at the bottom of extraction section 22.Examples of suitable mixing means 60 include low shear mixing devices,such as a turbine (e.g., Lightning Sealmaster P6X05E with an A310impeller operating at about 0–175 rpm), a marine impeller, a paddlemixer or an external recirculation loop having a low shear pump.

Vessel 10 further includes bottom tap 62, which extends from the lowerportion of extraction section 22. Bottom tap 62 is suitable for removingmicroparticles 11 and liquids, such as non-solvent, from vessel 10.Alternatively, dip tubes (not shown) may be used to removemicroparticles 11 and liquids from vessel 10.

When required for drug delivery, relevant internal portions of theapparatus of this invention are cleaned and sanitized, or sterilized,between each use to assure the sterility of the final product.

In the method of this invention, microparticles of a material are formedfrom a solution of the material in a suitable solvent. Materialssuitable for use in this method can include any soluble materials,provided a non-solvent is available which has a lower melting point thanthe solvent, and which has sufficient miscibility with the solvent toextract solid and/or thawed liquid solvent from a frozen microparticle.Preferably, materials used in this method include peptides,polypeptides, proteins, polymers, small molecule drugs and pro-drugs.

Any type of suitable polymer can also be used to form a microparticle.In a preferred embodiment, a polymer used in this method isbiocompatible. A polymer is biocompatible if the polymer, and anydegradation products of the polymer, such as metabolic products, arenon-toxic to humans or animals, to whom the polymer was administered,and also present no significant deleterious or untoward effects on therecipient's body, such as an immunological reaction at the injectionsite. Biocompatible polymers can be biodegradable polymers,non-biodegradable polymers, a blend thereof or copolymers thereof.

Suitable biocompatible, non-biodegradable polymers include, forinstance, polyacrylates, polymers of ethylene-vinyl acetates and otheracyl substituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, blends andcopolymers thereof.

Suitable biocompatible, biodegradable polymers include, for example,poly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s,poly(lactic acid)s, poly(glycolic acid)s, polycarbonates,polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters,polyacetals, polycyanoacrylates, polyetheresters, polycaprolactone,poly(dioxanone)s, poly(alkylene alkylate)s, polyurethanes, blends andcopolymers thereof. Polymers comprising poly(lactides), copolymers oflactides and glycolides, blends thereof, or mixtures thereof are morepreferred. Said polymers can be formed from monomers of a singleisomeric type or a mixture of isomers.

A polymer used in this method can be blocked, unblocked or a blend ofblocked and unblocked polymers. An unblocked polymer is as classicallydefined in the art, specifically having free carboxyl end groups. Ablocked polymer is also as classically defined in the art, specificallyhaving blocked carboxyl end groups. Generally, the blocking group isderived from the initiator of the polymerization reaction and istypically an alkyl radical.

Acceptable molecular weights for polymers used in this invention can bedetermined by a person of ordinary skill in the art taking intoconsideration factors such as the use of the microparticle, the desiredpolymer degradation rate, physical properties such as mechanicalstrength, and rate of dissolution of polymer in solvent. Typically, anacceptable range of molecular weights for polymeric microparticleshaving therapeutic uses is between about 2,000 Daltons to about2,000,000 Daltons.

In an even more preferred embodiment, the polymer is apoly(lactide-co-glycolide) with a lactide:glycolide ratio of about 1:1and a molecular weight of about 5,000 Daltons to about 70,000 Daltons.In an even more preferred embodiment, the molecular weight of thepoly(lactide-co-glycolide) used in the present invention has a molecularweight of about 5,000 Daltons to about 42,000 Daltons.

Typically, a suitable polymer solution contains between about 1% (w/w)and about 30% (w/w) of a suitable biocompatible polymer, wherein thebiocompatible polymer is typically dissolved in a suitable polymersolvent. Preferably, a polymer solution contains about 5% (w/w) to about20% (w/w) polymer.

Microparticles can be formed by either a continuous freezing andextraction process or by a batch process wherein a batch of frozenmicrodroplets is formed in a first step, and then in a separate secondstep, the frozen microdroplets in the batch are extracted to formmicroparticles.

In this method, freezing zone 26 includes the portion of freezingsection 20, which is substantially encircled by liquified gas flow 24.Freezing zone 26 is formed within freezing section 20 of vessel 10, bydirecting a flow 24 of a suitable liquified gas from at least two spraynozzles 32 in a substantially downward direction, toward side wall 12.Typically, the liquified gas discharged from spray nozzles 32 is angledsuch that the liquified gas impinges against side wall 12 to formliquified gas flow 24 along the inside surface of side wall 12, thuswetting side wall 12. In a preferred embodiment, liquified gas, fromeach of six spray nozzles 32, is directed against side wall 12 at anangle to side wall 12 of less than about 30° to reduce the splashing ordeflection of liquified gas off of side wall 12.

Alternately, liquified gas flow 24 is directed substantially parallelto, but displaced from the inside surface of side wall 12 to effectivelyform an independent wall of liquified gas extending from spray nozzles32 to inner wall 18.

Liquified gas is provided to spray nozzles 32 from liquified gas source36 through liquified gas inlet 34.

Liquified gases suitable for use in this method include liquid argon(−85.6° C.), liquid nitrogen (−95.8° C.), liquid helium or any otherliquified gas having a temperature sufficiently low to freezemicrodroplets 28 of a solution, while the microdroplets 28 are containedin freezing zone 26 or in liquified gas flow 24. Liquid nitrogen ispreferred.

In an alternate embodiment, illustrated in FIG. 2, freezing zone 24 isformed within freezing section 20, by directing liquified gas fromliquified gas source 36, through liquified gas inlet 34 and intoliquified gas space 104, wherein the liquified gas then flows up overweir 102, or through slots (not shown) in weir 102 to form liquified gasflow 24. Liquified gas flow 24 then flows substantially downward alongthe inside surface of weir 102.

Referring back to FIG. 1, microdroplets 28 of a solution, preferably asolution of a polymer, are then directed through freezing zone 26, in asubstantially downward direction, wherein microdroplets 28 freeze toform frozen microdroplets 44. A portion of microdroplets 28 may freezeby contacting liquified gas in liquified gas flow 24. Microdroplets 28were previously formed by directing the solution from solution source40, through solution inlet 38, into a suitable microdroplet formingmeans 30. Typically, within freezing section 20, at least a portion ofthe liquified gas will volatilize due to heat in-leakage and/or heattransfer from microdroplets 28 to the liquified gas.

A three-phase flow of volatilized gas, liquified gas and frozenmicrodroplets 44 then flows from the bottom of freezing section 20 andinto extraction section 22, through three-phase port 42.

In one embodiment, at least a portion of frozen microdroplets 44 areentrained within liquified gas flow 24, which then carry frozenmicrodroplets 44 into extraction section 22. The entrainment of frozenmicrodroplets 44 within liquified gas flow 24 may improve the finalyield of microparticles 11 produced, according to the method ofinvention, by transporting, into extraction section 22, frozenmicrodroplets 44 which might otherwise remain within freezing section20, such as by adhering to side wall 12 and/or internal wall 18, and/orby reducing the loss of airborne frozen microdroplets 44 from vessel 10through gas outlet 52.

The liquified gas is then separated from frozen microdroplets 44 bysuitable separating means, leaving frozen microdroplets 44 disposed inthe lower portion of extraction section 22.

In one embodiment, the liquified gas is heated to a temperature belowthe melting point of frozen microdroplets 44, but at or above theboiling point of the liquified gas whereby the liquified gas isvaporized and separated from frozen microdroplets 44.

Alternately, liquified gas can be separated by pulling a partial vacuumon extraction section 22 through gas outlet 52 and heating the liquifiedgas to a temperature below the boiling point of the liquified gas buthigh enough to elevate the vapor pressure of the liquified gas, therebyevaporating the liquified gas.

After heating, the liquified gas is volatilized, thereby separating theliquified gas from frozen microdroplets 44. The liquified gas can beheated by heat in-leakage from the outside environment through side wall12 and vessel bottom 16. Preferably, extraction section 22 is heated byan electrical heat source or by recirculating a warmer fluid, such asnitrogen gas or a nitrogen gas/liquified nitrogen mixture, through heatexchanger tubes 46. In addition, a fluid can be circulated, through heatexchanger tubes 46, to control temperature within extraction section 22to firstly volatilize the liquified gas in a controlled manner, and thensubsequently to slowly warm the solvent in frozen microdroplets 44 topermit solvent extraction into the liquid non-solvent.

Alternately, liquified gas is separated from frozen microdroplets 44 bydirecting the liquified gas through filter 50 and then out of extractionsection 22 through filtered bottom tap 48. Directing the liquified gasthrough filter 50 thereby removes the liquified gas from extractionsection 22, while retaining frozen microdroplets 44 within the bottomportion of extraction section 22.

Wherein the liquified gas is separated by heating to volatilize theliquified gas, the resulting volatilized gas is then directed out ofextraction section 22 through at least one gas outlet 52. Pressurewithin vessel 10 is primarily dependent upon the amount of liquifiedgas, which is volatilized within extraction section 22, and upon thedischarge rate of gas through gas outlet 52. Vessel 10 can be operatedat pressures above, equal to, or below atmospheric pressure. The upperpressure limit for performing this method is dependent upon the pressurerating of vessel 10.

It is preferred that the method of invention be performed, duringformation of frozen microdroplets 44, under a partial vacuum. Achievinga partial vacuum within extraction section 22, and thus throughoutvessel 10, is achieved by means known to one of skill in the art, suchas using a pump or blower to take a suction through gas outlet 52 onextraction section 22.

Following separation of frozen microdroplets 44 from the liquified gas,frozen microdroplets 44 are then contacted with a suitable cold liquidnon-solvent, which is at a temperature below the melting point of frozenmicrodroplets 44. In a preferred embodiment, the liquid non-solvent ismaintained below the melting point of frozen microdroplets 44, and thesolvent is extracted from the solid state into the liquid non-solvent toform porous microparticles 11 over a period of about 1 to about 24hours. The extraction of solid state solvent slows the extractionprocess, thereby providing greater control of extraction andmicroparticle 11 formation.

In another embodiment, the liquid non-solvent is warmed to a temperatureat or above the melting point of frozen microdroplets 44. The solvent infrozen microdroplets 44 thereby thaws and then, is extracted into thenon-solvent. The solvent is thereby extracted as a solid and/or a liquiddepending upon the various factors such as the volume of solvent infrozen microdroplet 44, the volume of non-solvent to which frozenmicrodroplet 44 is exposed, and the warming rate of frozen microdroplet44. Depending upon the warming rate, the microparticle produced can alsobe porous, for lower warming rates, or significantly less porous due topartial particle condensation following rapid solvent extraction.

Non-solvent can be in the form of a spray, a stream and/or extractionbath 56. Preferably, frozen microdroplets 44 are immersed within thenon-solvent of extraction bath 56.

Suitable non-solvents are defined as non-solvents of the material insolution, which are sufficiently miscible with the solvent of thesolution to extract said solvent, out of the frozen microdroplets 44 asthe solvent warms, thereby forming microparticles 11. In addition, thenon-solvent has a melting point below the melting point of the frozenmicrodroplets 44.

In another embodiment, second non-solvents, such as hexane, are added tothe first non-solvent, such as ethanol, to increase the rate of solventextraction from certain polymers, such as poly(lactide-co-glycolide).

In a preferred embodiment, at least a portion of frozen microdroplets 44are entrained within the non-solvent, which may improve the final yieldof microparticles 11 produced, according to the method of invention, bytransporting frozen microdroplets 44 into extraction bath 56. The frozenmicrodroplets may otherwise have been lost in the process due toadhering to side wall 12, and/or from the loss of airborne frozenmicrodroplets 44 from vessel 10 through gas outlet 52.

In a further embodiment, frozen microdroplets 44 are then agitatedwithin extraction bath 56 by mixing means 60 to reduce the concentrationgradient of solvent within the non-solvent surrounding each frozenmicrodroplet 44 or microparticle 11, thereby improving the effectivenessof the extraction process.

In yet another embodiment, the extraction process includes thesequential addition to, and drainage of separate aliquots of non-solventfrom, extraction section 22, to extract solvent into each separatealiquot. Extraction is thereby performed in a step-wise manner. Thawingrate is dependent on the choice of solvents and non-solvents, and thetemperature of the non-solvent in extraction section 22. Table 1provides exemplary polymer/solvent/non-solvent systems that can be usedin this method along with their melting points.

TABLE 1 Appropriate Polymer Solvents and Non-Solvents Systems, withSolvent and non-Solvent Melting Points POLYMER SOLVENT (° C.)NON-SOLVENT (° C.) Poly(lactide) Methylene Ethanol (−114.5) Chloride(−95.1) Chloroform (−63.50) Methanol (−97.5) Poly(lactide-co- EthylEthanol (−114.5) glyco-lide) Acetate (−83.6) Acetone (−95.4) Ethyl ether(−116.3) Methylene Pentane (−130) Chloride (−95.1) Isopentane (−160)Poly(capro- Methylene Ethanol (−114.5) lactone) Chloride (−95.1) Poly(vinyl Water (0) Acetone (−95.4) alcohol) Ethylene-vinyl MethyleneEthanol (−114.5) acetate Chloride (−95.1)

For proteins it is preferred that frozen microdroplets 44 be slowlythawed while the polymer solvent is extracted to produce amicroparticle.

A wide range of sizes of microspheres can be made by varying the dropletsize, for example, by changing the nozzle diameter or air flow into anair atomizer. If very large diameters of microparticles 11 are desired,they can be extruded through a syringe directly into freezing zone 24.Increasing the inherent viscosity of the polymer solution can alsoresult in an increasing microparticle size. The size of microparticles11 produced by this process can range from greater than about 1000 downto about 1 micrometer, or less, in diameter. Usually, a microparticlewill be of a size suitable for injection into a human or other animal.Preferably, the diameter of a microparticles 11 will be less than about180 micrometers.

Following extraction, microparticles 11 are filtered and dried to removenon-solvent, by means known to one of skill in the art. For a polymericmicroparticle, said microparticle is preferably not heated above itsglass transition temperature to minimize adhesion betweenmicroparticles, unless additives, such as mannitol, are present toreduce adhesion between the microparticles.

In another embodiment, a solution of a material also contains one ormore additional substance, which is dispersed within the solution. Saidadditional substance is dispersed by being co-dissolved in the solution,suspended as solid particles, such as lyophilized particles, within thesolution, or dissolved in a second solvent, which is immiscible with thesolution, and is mixed with the solution to form an emulsion. Solidparticles, suspended in the solution can be large particles, with adiameter greater than 300 micrometers, or micronized particles with adiameter as small as about 1 micrometer. Typically, the additionalsubstance should not be soluble in the non-solvent.

Wherein the material comprises a polymer, the polymer solution containsat least one biologically active agent. Examples of suitable therapeuticand/or prophylactic biologically active agents include proteins, such asimmunoglobulin-like proteins; antibodies; cytokines (e.g., lymphokines,monokines and chemokines); interleukins; interferons; erythopoietin;hormones (e.g., growth hormone and adrenocorticotropic hormone); growthfactors; nucleases; tumor necrosis factor; colony-stimulating factors;insulin; enzymes; antigens (e.g., bacterial and viral antigens); andtumor suppressor genes. Other examples of suitable therapeutic and/orprophylactic biologically active agents include nucleic acids, such asantisense molecules; and small molecules, such as antibiotics, steroids,decongestants, neuroactive agents, anesthetics, sedatives,cardiovascular agents, anti-tumor agents, antineoplastics,antihistamines, hormones (e.g., thyroxine) and vitamins.

Examples of suitable diagnostic and/or therapeutic biologically activeagents include radioactive isotopes and radiopaque agents.

The microspheres made by this process can be either homogeneous orheterogeneous mixtures of the polymer and the active agent. Homogeneousmixtures are produced when the active agent and the polymer are bothsoluble in the solvent, as in the case of certain hydrophobic drugs suchas steroids. Heterogeneous two phase systems having discrete zones ofpolymer and active agent are produced when the active agent is notsoluble in the polymer/solvent, and is introduced as a suspension oremulsion in the polymer/solvent solution, as with hydrophilic materialssuch as proteins in methylene chloride.

The amount of a biologically active agent, which is contained in aspecific batch of microparticles is a therapeutically, prophylacticallyor diagnostically effective amount, which can be determined by a personof ordinary skill in the art taking into consideration factors such asbody weight, condition to be treated, type of polymer used, and releaserate from the microparticle.

In one embodiment, a controlled release polymeric microparticle containsfrom about 0.01% (w/w) to approximately 50% (w/w) biologically activeagent. The amount of the agent used will vary depending upon the desiredeffect of the agent, the planned release levels, and the time span overwhich the agent will be released. A preferred range of loading forbiologically active agents is between about 0.1% (w/w) to about 30%(w/w).

When desired, other materials can be incorporated into microparticleswith the biologically active agents. Examples of these materials aresalts, metals, sugars, surface active agents. Additives, such as surfaceactive agents, may also be added to the non-solvent during extraction ofthe solvent to reduce the possibility of aggregation of themicroparticles.

The biologically active agent can also be mixed with other excipients,such as stabilizers, solubility agents and bulking agents. Stabilizersare added to maintain the potency of the agent over the duration of theagent's release. Suitable stabilizers include, for example,carbohydrates, amino acids, fatty acids and surfactants and are known tothose skilled in the art. The amount of stabilizer used is based onratio to the agent on a weight basis. For amino acids, fatty acids andcarbohydrates, such as sucrose, lactose, mannitol, dextran and heparin,the molar ratio of carbohydrate to agent is typically between about 1:10and about 20:1. For surfactants, such as the surfactants Tween™ andPluronic™, the molar ratio of surfactant to agent is typically betweenabout 1:1000 and about 1:20.

In another embodiment, a biologically active agent can be lyophilizedwith a metal cation component, to stabilize the agent and control therelease rate of the biologically active agent from a microparticle, asdescribed, in co-pending U.S. patent application Ser. No. 08/279,784,filed Jul. 25, 1994, the teachings of which are incorporated herein byreference.

Solubility agents are added to modify the solubility of the agent.Suitable solubility agents include complexing agents, such as albuminand protamine, which can be used to control the release rate of theagent from a polymeric or protein matrix. The weight ratio of solubilityagent to biologically active agent is generally between about 1:99 andabout 20:1.

Bulking agents typically comprise inert materials. Suitable bulkingagents are known to those skilled in the art.

Further, a polymeric matrix can contain a dispersed metal cationcomponent, to modulate the release of a biologically active agent fromthe polymeric matrix is described, in co-pending U.S. Pat. No.5,656,297, filed May 3, 1994, to Bernstein et al. and in a co-pendingInternational Application designating the United States, PCT/US95/05511,filed May 3, 1995, the teachings of which are incorporated herein byreference.

In yet another embodiment, at least one pore forming agent, such as awater soluble salt, sugar or amino acid, is included in themicroparticle to modify the microstructure of the microparticle. Theproportion of pore forming agent added to the polymer solution isbetween about 1% (w/w) to about 30% (w/w). It is preferred that at leastone pore forming agent be included in a non-biodegradable polymericmatrix of the present invention.

FIG. 3 illustrates yet another embodiment of an apparatus of theinvention, suitable for performing the method of invention. Theapparatus of FIG. 3 has many of the same elements of FIG. 1 and likeelements are designated with like numerals. In this apparatus, freezingsection 20 is disposed within freezing vessel 202, and is substantiallyenclosed by side wall 12, vessel top 14 and freezing vessel bottom 204.Extraction section 22 is disposed, likewise, within extraction vessel206, and is substantially enclosed by, side wall 12 a, extraction vesseltop 208 and vessel bottom 16. Freezing vessel 202 is disposed generallyabove extraction vessel 206. Conduit 210 is disposed between freezingvessel 202 and extraction vessel 206. Conduit 210 includes conduit inlet212, disposed at or near freezing vessel bottom 204, and conduit outlet214, disposed at or near extraction vessel top 208. Conduit 210 providesthree-phase communication, specifically solids, liquids and gases,between freezing section 20 and extraction section 22.

Optionally, conduit 210 includes three-phase mixing means 216 for mixingthe three phases in the three-phase flow, whereby at least a portion offrozen microdroplets 44 contained in the gaseous phase will be capturedin the liquid phase, thereby increasing product yield by reducing theloss of frozen microdroplets 44 from venting gases through gas outlet52. Suitable three-phase mixing means 216 include a cascading baffle, orpreferably, one or more elements of a static mixer (e.g., Model #KMR-SAN; Chemineer, Inc.). A preferred three-phase mixing means 216provides a tortuous flow. More preferably, three-phase mixing means 216comprises a number of in-series static mixer elements sufficient tocreate turbulent flow, typically four elements.

In a further embodiment, solution source 40, includes mix tank 218,having a second mixing means (not shown) and fragmentation loop 222. Anymeans for mixing a solution, suspension or emulsion is suitable for asecond mixing means. High shear mixing is preferred for the secondmixing means.

Fragmentation loop 222 includes fragmentation inlet 224, which isdisposed at, or near, the bottom of dispersion tank 218, fragmentationoutlet 226, which is disposed at dispersion tank 218 generally elevatedabove fragmentation inlet 224. Fragmentation loop 222 also includesfragmentation means 228, which is disposed between fragmentation inlet224 and fragmentation outlet 226, and which reduces, or micronizes thesize of particles suspended in the material solution; and which formsfiner, better blended, emulsions of immiscible liquids. Suitablefragmentation means 228 include means capable of fragmenting a solid toa diameter between about 1 micrometer, or less, and about 10micrometers. Examples of suitable fragmentation means 228 includerotor/stator homogenizers, colloid mills, ball mills, sand mills, mediamills, high pressure homogenizers.

In an alternate embodiment, fragmentation occurs within mix tank 218 bythe use of disruptive energy, such as that provided by a sonic probe,high shear mixer or homogenizer.

The temperature of dispersion tank 218 and/or of fragmentation loop 222is typically controlled when containing proteins, or other heatsensitive materials, by means known in the art, to minimize proteindenaturing.

In a method illustrated in FIG. 3, the volatilized gas, liquified gasand frozen microdroplets 44 are directed from freezing section 20 andthrough conduit 210, which includes three-phase mixing means 216,preferably a four-element, or more, static mixer, to turbulently mix thethree phases and scrub frozen microdroplets 44, which were entrained inthe gas phase, into the liquified gas, thereby improving yield.

In another embodiment, a solution containing an additional substance,which is in solid form or which forms an emulsion with the solvent, isrecirculated through fragmentation means 228, such as a homogenizer, tomicronize the solid particles, preferably particulates of about 1–10micrometers in diameter, or to further blend the emulsion to formsmaller emulsion droplets.

Fragmentation is not required when the solution has no suspendedparticles, or when larger suspended particles are desired.

Alternately, the second mixing means can be used as a fragmentationmeans, such as when a high speed/high shear mixer is used for the secondmixing means.

FIG. 4 illustrates yet another embodiment of an apparatus of theinvention, suitable for performing the method of invention. Theapparatus of FIG. 4 has many of the same elements of FIGS. 1 and 3, andlike elements are designated with like numerals. This apparatus includesmultiple freezing vessels 202, each containing a separate freezingsection 20. The apparatus also includes one extraction vessel 206,having extraction section 22. Three-phase communication is provided fromeach freezing section 20 to extraction section 22 by separate conduits210. Each conduit 210 includes separate three-phase mixing means 216.

In the method illustrated in FIG. 4, frozen microdroplets 44 are formedin each freezing section and then transferred to a common extractionsection 22.

The composition made according to the method of this invention can beadministered to a human, or other animal, orally, by suppository, byinjection or implantation subcutaneously, intramuscularly,intraperitoneally, intracranially, and intradermally, by administrationto mucosal membranes, such as intranasally or by means of a suppository,or by in situ delivery (e.g. by enema or aerosol spray) to provide thedesired dosage of a biologically active agent based on the knownparameters for treatment of various medical conditions.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. A method for forming microparticles comprising the steps of: a)directing microdroplets of a mixture comprising a biocompatible polymer,a solvent for the polymer and a protein, peptide or small molecule, intoa freezing section containing a liquefied gas, whereby the microdropletsfreeze; and b) contacting the frozen microdroplets in an extractionsection with a liquid non-solvent to extract the solvent into thenon-solvent thereby forming said microparticles; wherein the freezingsection and extraction section are separated, and the non-solvent is inthe liquid state throughout the method.
 2. The method of claim 1,wherein the biocompatible polymer is biodegradable.
 3. The method ofclaim 2, wherein said biocompatible and biodegradable polymer isselected from the group consisting of poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,polycarbonates, polyesteramides, polyanhydrides, poly(amino acids),polyorthoesters, polycaprolactone, poly(dioxanone)s, poly(alkylenealkylate)s, polyurethanes, blends and copolymers thereof.
 4. The methodof claim 3, wherein the polymer is a poly(lactide-co-glycolide).
 5. Themethod of claim 1, wherein the temperature of step (b) is lower than thetemperature of step (c).
 6. The method of claim 1, wherein the liquefiedgas is sprayed into the freezing section.
 7. The method of claim 1,wherein the frozen microdroplets are collected at the bottom of thefreezing section and directed into the extraction section.
 8. A methodfor forming microparticles comprising the steps of: a) directing themicrodroplets of a mixture comprising a biocompatible polymer, a solventfor the polymer and a protein, peptide or small molecule, into afreezing vessel containing a liquefied gas, whereby the microdropletsfreeze; and b) contacting the frozen microdroplets in an extractionvessel with a liquid non-solvent to extract the solvent into thenon-solvent thereby forming said microparticles; wherein the freezingvessel and extraction vessel are separated, and the non-solvent is inthe liquid state throughout the method.
 9. The method of claim 8,wherein the biocompatible polymer is biodegradable.
 10. The method ofclaim 9, wherein said biocompatible and biodegradable polymer isselected from the group consisting of poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,polycarbonates, polyesteramides, polyanhydrides, poly(amino acids),polyorthoesters, polyacetals, polycyanoacrylates, polyetheresters,polycaprolactone, poly(dioxanone)s, poly(alkylene alkylate)s,polyurethanes, blends and copolymers thereof.
 11. The method of claim 8,wherein the polymer is a poly(lactide-co-glycolide).
 12. The method ofclaim 8, wherein the temperature of step (b) is lower than thetemperature of step (c).
 13. The method of claim 8, wherein theliquefied gas is sprayed into the freezing vessel.
 14. The method ofclaim 8, wherein the frozen microdroplets are collected at the bottom ofthe freezing vessel and directed into the extraction vessel.
 15. Themethod of claim 1, wherein the protein, peptide or small molecule isdissolved in the mixture.
 16. The method of claim 1, wherein theprotein, peptide or small molecule is suspended in the mixture.
 17. Themethod of claim 1, wherein the protein, peptide or small molecule formsan emulsion in the mixture.
 18. The method of claim 8, wherein theprotein, peptide or small molecule is dissolved in the mixture.
 19. Themethod of claim 8, wherein the protein, peptide or small molecule issuspended in the mixture.
 20. The method of claim 8, wherein theprotein, peptide or small molecule forms an emulsion in the mixture.